VENEZUELAN EQUINE ENCEPHALITIS VIRUS ACTIVITY IN NORTHERN COLOMBIA DURING APRIL AND MAY 1983l

R. Its: Dickerman, E. W, C~lpp,~ H. Gro~.t,~ A. MoraZes AZarcon, 5 E. Cnra, 6 A. K Dickerman, 7 A, L. I&agos, 6 R. R&co-Hesse, 7 C. A. ByZor, 7 and S. C. Weaver7

I N’I’RODUCTION drainage area that have isolated VEE virus have previously been reported (I, 2). We The source of equine-virulent report here the isolation and identifica- strains of Venezuelan equine encephalitis tion of strains of VEE virus from sentinel (WE) virus that sporadically cause epi- hamsters exposed in an extensive region demics and equine epizootics is un- of tropical wet forest in Colombia adja- known. This is the most important un- cent to the Venezuelan border, as well as answered scientific and public health data on mosquito collections frorn sev- question relating to VEE virus. One possi- eral foci of VEE virus. This investigation bility is that epizootic virions exist at low was conducted as a joint program be- levels in populations of enzootic strains. tween Cornell University in Ithaca, New These virions could then break out into a York, USA, and Colombia’s National In- visible epizootic cycle through the com- stitute of Health (Instituto National de bination of chance selection and fortui- Salud) in Bogota. tous passage through susceptible mam- mals, including equine animals and man, that produce high levels of viremia capable of infecting inefficient vectors. Arbovirus studies in north- western Venezuela’s Lake Maracaibo ) Associate Professor, Department of Entomology, Cor- nell University, Ithaca, New York 14853, USA. 4 Director, Virology Section, Instituto National de Sa- lud, Bogota, Colombia. ) Director, Entomology Section, Instituto National de Salud, Bogota, Colombia. I This article will also be published in Spanish in the Bo- 6 Research Scientist, Entomology Section, Instituto Na- letin de ia Oficna Sanitatia Panamericana, 1987. The cional de S&d, Bogota. Colombra. investigation reported here was carried out in collabora- ’ Research Assistant, Cornell University Medical College, tion with the Pan American Health Organization and New York, New York, USA. was supported in part by National Science Foundation Cooperative Science Program Grant NSF-INT 8203076. z Associate Professor, Department of Microbiology, Cor- nell University Medical College, 1300 York Avenue, New York, New York 10021, USA. from a sentinel hamster exposed near M ATERIALS Tibu in the Rio de Oro region in 1970 AND METHODS (Groot, personal communication). Field and Laboratory Techniques Study Areas Sentinel hamsters. Outbred Syrian Santa Matta region. Sentinel hamsters hamsters from the colonies of the Insti- were exposed at two forests, “Guachaca” tuto National de Salud (INS) were ex- and “Don Diego,” that are respectively posed in sentinel cages as described pre- located 44 and 61 kilometers east of the viously for Middle America (4) or in port of Santa Marta (see Figure 1). We plastic “coquito” cages. The latter were found Guachaca to be a swamp forest originally designed by Karl M. Johnson with abundant standing water, whereas (then at the Middle American Research the tropical wet forest encountered at Unit, Panama) and modified and pro- Don Diego had well-drained soil, with duced at the INS. They were made of water standing only in some drainage two-liter plastic mixing bowls with tight channels. VEE virus was isolated in the re- snap-on covers. Most of the plastic of the gion from CtiZex (Mehnoconion) mos- cover was removed and replaced with quitoes during the period October 1973- half-inch metal mesh to form the floor of July 1974 and from sentinel hamsters in the cage when the bowl was suspended December 1975 and March and April upside down from a hook inserted 1976 (Groot, personal communication). through the reinforced bottom of the bowl. The snap-on cover was easily re- Magangue region. Sentinel hamsters moved later, permitting easy handling of were exposed in a low, flood-plain forest the animal. This design also provided approximately two kilometers northwest ready accessto the animal for mosquitoes of Boquillas on a side-channel of the seeking a blood meal. Magdalena River, about 30 kilometers In addition, 14 hamsters were southeast of Magangue. The water table exposed in Trinidad No. 10 mosquito was high, and standing water was abun- traps (j) to limited numbers of mosqui- dant. The “Magangue” strain of VEE was tos at Guachaca (an average of 2.3 per identified in this region in 1969 (3). hamster-night), Don Diego (an average of 2 .s) and Rio de Oro (an average of Rio de Oro region. A large area of trop- 3.5). Only the two hamsters exposed in ical wet forest investigated along the Rio No. 10 traps at Rio de Oro were tested de Oro was contiguous with the “Can0 for virus. Mocha” site in adjacent Venezuela (2). Hamsters were exposed in dense virgin Collection, processing, and testing of forest about two kilometers northeast of specimens from vertebrates. Ran- the village of Rio de Oro. One strain of domly selected human subjects were VEE virus had previously been isolated bled by regional public health personnel using standard venipuncture techniques at Magangue and Rio de Oro. Equine an- imals, wild birds, and mammals (includ- ing bats) were captured and bled as de- scribed previously (6). The procedures 2!77 FIGURE1. A map of Colombiaand Venezuelashowing the study areas.

I

COLOMBIA

VENEZUELA used to processthese blood specimens for serology and hamster tissues for virus iso- lation, as well as the techniques used for ICEsums virus isolation, have previously been re- Virus Isolation and Identification ported (6, 7). The vaccine strain of VEE virus (TC-83) was used in plaque-reduc- There were no fatalities tion neutralization tests conducted with among the 52 hamsters exposed in tropi- primary chick embryo cell (CEC) cultures cal wet forests near Santa Marta for a to- for testing human and equine sera. A tal of 515 hamster-nights (Table 1). Fif- l%o neutralization index of 1.6 or teen of 27 hamsters exposed near greater was considered positive. Poly- Boquillas in the Magangue region were valent VEE virus mouse ascitic fluid made removed from the field moribund or by W. F. Scherer in 1966 against 12 dead. However, none yielded a plaque- strains of subtype I-E virus from Mexico forming virus in chick embryo cell cul- was used in virus identification neutrah- tures. Seven of the tissue suspensions zation tests. were also tested by intracranial inocula- tion into suckling mice; all of these tests Mosquito collections. Adult mosquito yielded negative results. collections were made within and proxi- At Rio de Oro, 36 of the 37 mal to forested sites near Rio Guachaca, hamsters exposed in sentinel hamster 3 Rio Don Diego, and Rio de Oro using a cages or in Trinidad No. 10 mosquito D-Vat vacuum sweeper and CDC minia- traps were removed from the field mori- 3 ture light traps or human hosts as attract- bund or dead. Tissues from 19 of these 3 ants. Female mosquitoes were pinned 37 hamsters (including both hamsters and shipped to the Department of Ento- from the mosquito traps) were tested for 2q mology at Cornell University for identifi- virus; all yielded plaque-forming virus in cation; CaZex(Mehmconion) spp. males chick embryo cell cultures that was neu- E were dissected, and their genitalia were tralized by VEE virus-specific antibody. cleared and mounted on slides for later Of the 18 hamsters not tested, 10 were s identification. replacements put into cageswhere senti- nel hamsters had died previously, and so 2 Hydroxylapatite chromatography. their infections may have been caused by p The methods of Jahrling and Eddy (s) focally recycled virus. Six animals were i=: were employed, except that virus was ini- spoiled when recovered from the field 2 tially washed from the column with a (see discussion), and two tissue samples 3 0.15 M phosphate solution at pH 7 .O be- were not found after returning to New !s fore elutions with the phosphate gradi- York. The average number of hamster- ent were begun. Virus in the fractions nights exposure for animals that were s! was assayedfor plaque-forming units. probably not infected with focally recy- . cled virus was 7.7. VEE virus isolations per -i 100 hamster-nights among this latter z group of animals was 13. B Serology in Humans and Animals $ 42 at Boquillas Q No VEE virus was isolated from the tissues of 15 dead or moribund 279 TABLE1. The numbers of hamstersexposed at four endemicfoci of Venezuelanequine encephalitisvirus in Colombiain 1983, together with the numbers becomingmoribund or dying and the strains of virus isolated,by locality.

No. of No. of No. of hamsters Hamsters-nights No. becoming No. hamsters strains of VEE Locality exposed of exposure ill or dyingI exposed testedI virus isolated Santa Marfa Region: Guachacaforest 22 279 o/22 Nonetested Don Diegoforest 32 236 Z/32 Nonetested MagangueRegion: Boquillas 27 231 IV27 15/O Tibu Region: Rio de Oro 25 (12)a 164 (90)a 36/37 19/19b a The numbers in parenthesisare the number of hamstersexposed and the number hamster-nightsof exposurefor animals moved to sites where a sentinel had died previously Viral isolates fmm these animals could representfocally recycledvirus b The denominatorincludes two Isolatesthat may represent recycledvirus (as explainedin footnotea above) and one isolatefrom the tissues of a seemingly heaithy hamsterthat was killed at the end of the study hamsters exposed near Boquillas. How- bait at Rio Guachaca differed markedly ever, 12 equine animals 2 to 10 years of from the species composition of the day- age were sampled along the river near the time collections. CzZex (MeZ.) qbda was forest where the hamsters were exposed, the predominant taxon, followed by Cx. and 11 of these were positive for VEE vi- (Cx.) species (principally Cx. nig@aZpus rus-specific neutralizing antibodies. and a few Cx. comziger) and by Ae. Twelve people 25 to 67 years of age (the f%Zvzuf%Zvus. D-Vat collections also con- average age being 47.9 years) were also tained numerous males later identified positive for VEE neutralizing antibodies, as Cx. (Med.) idottus. Large numbers of while 23 people 1 through 64 years old Deinocerites mosquitoes (near De. atdan- (with an average age of 26.7 years) ticzcs)were also collected in the vacuum yielded negative results. sweeper by selectively sampling crab- holes. Mosquito Collections Though not as abundant be- cause of drier conditions, the mosquito About 3,600 mosquitoes were fauna at Rio Don Diego was more diverse collected at the Rio Guachaca and Rio with regard to Cx. GyMa, Cx. spissipes, Don Diego sites, 60% of these coming and Cx. pedroi. As in the caseof the Rio from daytime collections made with hu- Guachaca collections, Ps. ferox was the man bait. Psoropbora feTox was found to predominant diurnally active species. be the predominant diurnally active spe- Czllex pedroi was a common cies at both locations. Females of this anthropophilic species found in the wet speciesreadily attacked at the edge of the forest habitat near Rio de Oro. This mos- forest as well as along trails leading in- quito was readily attracted to humans at to the interior, Aedes fuZvvus f~dvm, Ae. angzlstivi$ta$as, Ae. scapuZani, and members of the Ae. ternens group (near Ae. whitmorei) were collected in fewer numbers within the forest proper. The species composition of twilight collections made with human dusk and was also abundant in light-trap regions after lapses of 9 and 16 years, re- collections. Specifically, seven twilight spectively, despite “adequate” exposure collections made on successive evenings periods exceeding 200 hamster-nights. with human bait yielded collections of In view of the perpetuation of VEEvirus C&x (Mehnoconion) mosquitoes that at other relatively unmodified sites over consisted 53 % to 86 % of Cx. pedroi, the similar time spans, such as sites in the average percentage of Cx. ped-oi being Tibu region of Colombia or at La Ave- 68%. Cdex spissz$eswas also taken fre- llana in Guatemala (9, lo), the failure to quently in both types of collections but isolate virus in the Santa Marta and Ma- in considerably fewer numbers. As at the gangue regions during this investigation two sites near Santa Marta, Ps. ferox was must be considered a chance phenome- again the predominant day-biting mos- non. This failure does not prove the dis- quito, far outnumbering any other spe- appearance of the virus, but perhaps re- cies within the forest habitat. lates to local conditions-especially in the Magangue region, where children Hydroxylapatite Elution Profiles and young animals possessedVEE virus- of Rio de Oro VEE Strains specific neutralizing antibodies. The findings thus demonstrated further the The elution profiles were de- need for more frequent long-term moni- termined for three strains: 83U18, toring of enzootic situations. 83U78, and 83U434. Two strains eluted The easily constructed, light- at a phosphate molarity of about 0.15, weight, compact, and stackable “co- while more than 90 % of the third strain quite” sentinel hamster cages provided (83U78) eluted at a phosphate molarity an inexpensive and utilitarian field tool. of lessthan 0.20. These three strains were On the basis of our experience, minor therefore considered to represent an en- modifications are recommended to fur- zootic variety, supposedly lD, rather ther improve their design-specifically, than an epizootic variety-which would to prevent the retention of urine in the have eluted at a molarity in the range of groove where the cage and the floor 2.0-3.0 (8). meet. We found that the soiled cagesat- tracted flies, which ovideposited on the moribund sentinels and caused rapid spoilage. D ISCUSSION Findings from the vector stud- AND CONCLUSIONS ies, while principally qualitative, did not These studies demonstrate demonstrate any major differences be- the continued existence of VEE virus in tween the mosquito faunas at the sites enzootic foci within tropical wet forests near Santa Marta and those at Rio de Oro in northeastern Colombia along the Ven- in the Tibu region. Of the species col- ezuelan border in the Lake Maracaibo lected at Rio Guachaca that have been drainage area. VEEvirus activity has been circumstantially associated with the demonstrated periodically in this area transmission of enzootic VEEviruses, only since 197 1, when the Tibu strain was iso- lated. These records antedate by two years similar findings in the Catatumbo region of northwestern Venezuela (1, 2). In contrast, VEEvirus was not isolated in the Santa Marta or Magangue CX. cqybda was noteworthy. This species has been placed in the Spissipes section CKNOWLEDGMENTS of Ctidex (Melanoconion), a taxonomic A grouping that contains several proven We wish to acknowledge our vectors (11). legacy to our colleague and mentor, Dr. Interestingly, the forest near William F. Scherer, for his enthusiasm Rio Don Diego contained four species and enjoyment of field research and belonging to the Spissipes section (Cx. training and his fundamental contribu- adamesi, Cx. crybdu, Cx. spiss$es, and tion to the development of this program. Cx. pedroz). Based on previous studies in The authors also wish to express their Panama (revised by Galindo, I2), all gratitude to Gilbert0 Tones, Jose Mufioz, should be considered potential vectors. Jose Botero, and Indalesio Amaya for Of these, Cx. pedroi would be especially their expert assistance in the field work; suspect because it is very closely related to Doiia Caste110for her excellent techni- ecologically and taxonomically to Cx. cal assistance in the laboratory; and to taeniopzls (13), a widespread enzootic Dr. Sunthorn Sirivanakarn for confirm- VEEvector in Middle America (14). CE ing the identification of several CaZex Zex pedroi in particular, as well as Cx. (MeZmoconion) species. spissipes, were also abundant in light- trap and human bait collections made at Rio de Oro, where virus transmission to sentinel hamsters occurred readily. SUMMARY The role of Ps. ferox as an en- zootic VEE vector in Colombia is prob- Venezuelan equine encepha- litis virus was isolated from sentinel ham- lematic. While this speciescannot be dis- missed-because of earlier laboratory sters exposed in the Rio de Oro region of studies suggesting its susceptibility to Colombia, in the Lake Maracaibo drain- age area near the border with Venezuela, VEE virus infection (15) and its pro- in 1983-l 3 years after the first strain nounced abundance at each site-it is in- teresting to note that Ps. ferox did not was isolated from the region near Tibu. Hydroxylapatite elution pro- yield a single VEE virus isolate in the Ca- files of three isolates from the Rio de Oro tatumbo region of Venezuela during an region were typical of enzootic strains. eight-year study (2). Therefore, until fur- Relative abundance data and past isola- ther controlled laboratory and field stud- tions suggest that CzlGex(MeZanoconion) ies can be conducted, this species should pedroi is a probable enzootic vector at continue to be considered merely a po- this focus. tential vector. The virus was not isolated from forests near Santa Marta and Ma- gangue in the Rio Magdalena drainage area in 1983-9 and 16 years, respec- tively, after the last previous isolations in those areas. However, equine animals 2 to 10 years of age and humans 25 to 67 years of age living near the Magangue field study area were positive for VEE vi- t-us-specific neutralizing antibodies. 282 8 Jahrling, P B., and G. A. Eddy. Comparisons

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